Enhancing Activity of CAR T Cells by Co-Introducing a Bispecific Antibody

ABSTRACT

The present invention provides compositions and methods for treating cancer in a human. The invention includes administering a T cell, genetically modified to express a chimeric antigen receptor (CAR), a bispecific antibody, or a combination thereof to a subject. The CAR and bispecific antibody of the invention can comprise a human antibody, a humanized antibody, or antigen-binding fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/910,837, filed Jun. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/690,383, issued as U.S. Pat. No. 10,696,749, adivisional of U.S. patent application Ser. No. 14/410,427, filed Dec.22, 2014, issued as U.S. Pat. No. 9,765,156, a U.S. national phaseapplication filed under 35 U.S.C. § 371 claiming benefit toInternational Patent Application No. PCT/US2013/050275 filed on Jul. 12,2013, which is entitled to priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/671,535, filed Jul. 13, 2012, each ofwhich is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The Sequence Listing submitted herewith as a xml file named“046483-6073US4(03293) Sequence Listing ST_26,” created on Sep. 14, 2023and having a size of 62,746 bytes, is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

The development of T cells which are genetically modified to express achimeric antigen receptor (CAR) has opened the door for many newpotential therapies for cancers and other disorders. Generally, CARscomprise an extracellular antigen recognition domain and anintracellular domain.

Some CARs utilize a non-human extracellular antigen recognition domain,which can introduce an unwanted immune response that can jeopardize thetherapeutic benefits of CAR mediated tumor recognition and tumor lysis.For example, patients can react to mouse-derived antibodies and createantibodies that are specific to the foreign mouse-derived antibody. Thisresponse, termed the human anti-mouse antibody (HAMA) response, cangenerate symptoms similar to an allergic reaction that ranges from amild rash to life-threatening complications. Therefore, CARs thatcomprise an antigen recognition domain derived from human or humanizedantibodies may be beneficial because they would not stimulate such ahazardous immune response.

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that bindto a T cell antigen (e.g. CD3) and a tumor antigen. BiTEs have beenshown to induce directed lysis of target tumor cells and thus alsoprovide great potential therapies for cancers and other disorders.However, systemic delivery of BiTEs can result in toxicity, andtherefore more directed delivery of BiTEs to a specific tumorenvironment may be desirable.

Thus, there is an urgent need in the art for compositions and methodsfor treatment of cancer using human or humanized CARs and for thedirected delivery of therapeutic bispecific antibodies. The presentinvention satisfies this unmet need.

SUMMARY OF THE INVENTION

The invention provides an isolated nucleic acid sequence comprising asequence encoding a chimeric antigen receptor (CAR), wherein the CARcomprises an antigen binding domain derived from a bispecific antibody,a transmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from the group consisting of ahuman antibody, a humanized antibody, an antigen binding fragmentthereof, and any combination thereof.

In one embodiment, the isolated nucleic acid sequence encoding a CARcomprises the nucleic acid sequence selected from the group consistingof SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

In one embodiment, the antigen-binding fragment is a Fab or a scFv.

In one embodiment, the antigen binding domain binds to a tumor antigen.

In one embodiment, the tumor antigen is associated with a hematologicmalignancy.

In one embodiment, the tumor antigen is associated with a solid tumor.

In one embodiment, the tumor antigen is selected from the groupconsisting of CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met,PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and anycombination thereof.

In one embodiment, the CAR further comprises a costimulatory signalingregion comprising the intracellular domain of a costimulatory moleculeselected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and anycombination thereof.

In one embodiment, the isolated nucleic acid sequence encoding a CARcomprises a sequence encoding a bispecific antibody.

In one embodiment, the nucleic acid sequence encoding the bispecificantibody comprises the nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, and any combination thereof.

In one embodiment, the bispecific antibody comprises a human antibody,or an antigen-binding fragment thereof.

In one embodiment, the bispecific antibody comprises a humanizedantibody, or an antigen-binding fragment thereof.

The invention provides a cell comprising a nucleic acid sequencecomprising a sequence encoding a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain derived from abispecific antibody, a transmembrane domain, and a CD3 zeta signalingdomain, further wherein the antigen binding domain is selected from thegroup consisting of a human antibody, a humanized antibody, an antigenbinding fragment thereof, and any combination thereof.

In one embodiment, the cell is a T cell.

In one embodiment, the cell exhibits an anti-tumor immunity when theantigen binding domain binds to its corresponding antigen.

The invention provides a method for stimulating a T cell-mediated immuneresponse to a target cell population or tissue in a mammal, the methodcomprising administering to a mammal an effective amount of a cellgenetically modified to express a CAR and a bispecific antibody, whereinthe CAR comprises an antigen binding domain, a transmembrane domain, anda CD3 zeta signaling domain, further wherein the antigen binding domainis selected from the group consisting of a human antibody, a humanizedantibody, an antigen binding fragment thereof, and any combinationthereof, thereby stimulating a T cell-mediated immune response to atarget cell population or tissue in the mammal.

The invention provides a method for stimulating a T cell-mediated immuneresponse to a target cell population or tissue in a mammal, the methodcomprising administering to a mammal an effective amount of a cellgenetically modified to express a CAR, wherein the CAR comprises anantigen binding domain derived from a bispecific antibody, atransmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from a human antibody, ahumanized antibody, an antigen binding fragment thereof, and anycombination thereof, thereby stimulating a T cell-mediated immuneresponse to a target cell population or tissue in the mammal.

The invention provides a method of providing an anti-tumor immunity in amammal, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR and a bispecificantibody, wherein the CAR comprises an antigen binding domain, atransmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from a human antibody, ahumanized antibody, an antigen binding fragment thereof, and anycombination thereof, thereby providing an anti-tumor immunity in themammal.

The invention provides a method of providing an anti-tumor immunity in amammal, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR, wherein the CARcomprises an antigen binding domain derived from a bispecific antibody,a transmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from a human antibody, ahumanized antibody, an antigen binding fragment thereof, and anycombination thereof, thereby providing an anti-tumor immunity in themammal.

The invention provides a method of treating a mammal having a disease,disorder or condition associated with an elevated expression of a tumorantigen, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR and a bispecificantibody, wherein the CAR comprises an antigen binding domain, atransmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from a human antibody, ahumanized antibody, an antigen binding fragment thereof, and anycombination thereof, thereby treating the mammal.

The invention provides a method of treating a mammal having a disease,disorder or condition associated with an elevated expression of a tumorantigen, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR, wherein the CARcomprises an antigen binding domain derived from a bispecific antibody,a transmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from a human antibody, ahumanized antibody, an antigen binding fragment thereof, and anycombination thereof, thereby treating the mammal.

The invention provides a method of treating a human with cancer, themethod comprising administering to the human a cell geneticallyengineered to express a CAR and a bispecific antibody, wherein the CARcomprises an antigen binding domain, a transmembrane domain, and a CD3zeta signaling domain, further wherein the antigen binding domain isselected from a human antibody, a humanized antibody, an antigen bindingfragment thereof, and any combination thereof, wherein the cell is a Tcell.

The invention provides a method of treating a human with cancer, themethod comprising administering to the human a cell geneticallyengineered to express a CAR, wherein the CAR comprises an antigenbinding domain derived from a bispecific antibody, a transmembranedomain, and a CD3 zeta signaling domain, further wherein the antigenbinding domain is selected from a human antibody, a humanized antibody,an antigen binding fragment thereof, and any combination thereof,wherein the cell is a T cell.

The invention provides an isolated nucleic acid sequence encoding abispecific antibody, wherein the nucleic acid sequence comprises thenucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

The invention provides a cell comprising a nucleic acid sequenceencoding a bispecific antibody, wherein the nucleic acid sequencecomprises the nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

The invention provides a method for stimulating a T cell-mediated immuneresponse to a target cell population or tissue in a mammal, the methodcomprising administering to a mammal an effective amount of a cellgenetically modified to express a bispecific antibody.

The invention provides a method of providing an anti-tumor immunity in amammal, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a bispecific antibody.

The invention provides a method of treating a mammal having a disease,disorder or condition associated with an elevated expression of a tumorantigen, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a bispecific antibody.

The invention provides a method of treating a human with cancer, themethod comprising administering to the human a cell geneticallyengineered to express a bispecific antibody.

In one embodiment, the cell genetically engineered is an autologous Tcell.

In one embodiment, the cell secretes the bispecific antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts vector maps of RNA constructs. In vitro transcription(IVT) vector maps for Blinatumomab (pGEM-Blina.64A), CARs with4-1BB-zeta signaling domains against human CD19 fully human Ab 21D4(pGEM.D4(CD19).BBZ.64A), humanized HB12B (pGEM.12B(CD19).BBZ.64A) andmouse origin CD19 scFv from Blinatumomab (pGEM.Blina. 19BBZ.64A).

FIG. 2 is a chart depicting the experimental design for testing BlinaRNA electroporated CD19 CAR T cells. Day 10 stimulated T cells (fromND200) were electroporated as indicated. EP#1 with 10 μg FCM63 CD19 BBZRNA, EP#2 with 5 μg FCM63 CD19 BBZ RNA, EP#3 with 5 μg FCM63 CD19 BBZplus 10 μg Blina RNA, EP#4 with 10 μg Blina RNA and EP#5 with 5 μg GFPRNA. #12 was T cell without electroporation. Immediately afterelectroporation, an aliquot of equal volume (1 ml) of the electroporatedT cells were pooled as indicated for #6 to #8. Fifteen hours postelectroporation, another aliquot (1 ml) of electroporated cells werepooled as indicated for #9 to #11 and subsequently, the cells weresubjected to FACS staining for CAR detection, CD107a assay and cytotoxicT lymphocyte (CTL) assay.

FIG. 3 is a series of graphs depicting CAR staining of CD19 CAR, BlinaRNA electroporated T cells and GFP RNA electroporated T cells incubatedwith Blina RNA electroporated T cells. Fifteen hours afterelectroporation, the T cells for all the treatments as shown in FIG. 2were stained with a goat anti-mouse Fab. The number shown above eachplot refers to the conditions listed in FIG. 2 . The results show thatnot only could CD19 CAR RNA electroporated T cells be stained for CARexpression, but the Blina bis-RNA alone (#4) electroporated, and the GFPRNA electroporated T cells that were co-incubated with T cells that hadbeen electroporated with Blina bis-RNA (#7, #8, #10, #11) could also bestained. This demonstrates that the CD19-CD3 bispecific Ab secreted byBlina electroporated T cells could bind to CD3 of GFP electroporated Tcells.

FIG. 4 is a series of graphs depicting the results of experimentsillustrating that T cells electroporated with Blina bis-RNA “BITE” couldspecifically recognize tumors. Fifteen hours after electroporation, Tcells were subjected to a CD107a degranulation assay. Fifteen hoursafter electroporation, the T cells for all the conditions shown in FIG.2 were co-cultured with different types of cells that express CD19(Nalm6, K562-CD19 and Raji) or the CD19 negative tumor K562. The numbershown above each plot are the conditions listed in FIG. 2 . The resultsdemonstrate that T cells electroporated with Blina bis-RNA specificallyrecognized CD19 positive tumors.

FIG. 5 is a series of graphs depicting the results of experimentsdemonstrating that T cells electroporated with Blina “BITE” bispecificanti-CD19/CD3 RNA could enable non-tumor reactive, GFP RNAelectroporated T cells to specifically recognize tumors. Fifteen hoursafter electroporation, the T cells for all the conditions shown in FIG.2 were co-cultured with different cells that express CD19 (Nalm6,K562-CD19 and Raji) or the CD19 negative tumor K562 for CD107adegranulation assay. The number shown above each plot are the conditionslisted in FIG. 2 . The results show that T cells electroporated withBlina bis-RNA specifically recognized tumors. The results show that Tcells electroporated with Blina bis-RNA could enable non-tumor reactive,GFP RNA electroporated “bystander” T cells to specifically recognizetumors (#7, #8, #10, #11). CD8+T cells were gated.

FIG. 6 is a graph depicting the results of experiments illustrating thatT cells electroporated with Blina bis-RNA specifically kill CD19 antigenexpressing cells. Fifteen hours after electroporation, the T cells forall the conditions shown in FIG. 2 were tested for their lytic activityin a flow based CTL assay using K562-CD19-CFSE/K562-meso-CMRA as targetcells. The number shown for each condition of electroporated T cells arethe numbers listed in FIG. 2 . The results show that co-electroporationof Blina bispecific anti-CD19/anti-CD3 RNA with CD19 CAR RNA couldfurther enhance the killing activity of the T cells (#3 versus #2).Adding an equal number of non-tumor reactive, GFP electroporated T cellsto CD19 CAR RNA electroporated T cells reduced the killing ability ofCD19 CAR expressing T cells by reducing E:T ratio, while adding an equalnumber of non-tumor reactive, GFP electroporated T cells to Blinabis-RNA electroporated T Cells (#7, #8, #10, #11) could maintain T cellkilling activity equivalent to their non-diluted groups (#3 and #4),indicating that anti-CD19/anti-CD3 bispecific Ab secreted by Blinabis-RNA electroporated T cells could bind to GFP RNA electroporated Tcells and efficiently kill the target cells, thus effectivelymaintaining the E:T ratio.

FIG. 7 is a series of graphs depicting the staining of CAR expression ofnew CD19 CAR RNA electroporated RNAs. The expression of the newlyconstructed CD19 CARs is shown fifteen hours after electroporation of Tcells as indicated. Two different anti-IgG Fab were used: Anti-mIgG Fabwas used to stain mouse derived CAR, and anti-hIgG Fab was used to stainhuman derived CARs.

FIG. 8 is a series of graphs depicting the results of experimentsillustrating that both CD19 CARs from fully human Ab 12D4 and humanizedHB12B specifically recognize CD19 positive tumors. Fifteen hours afterelectroporation, the electroporated T cells as indicated wereco-cultured with two CD19 positive tumor lines (K562-CD19 or Raji) andone CD19 negative line U266B1 for a CD107a degranulation assay. Comparedwith currently used FMC63 CD19 CAR (10OF), both CD19 CARs from fullyhuman Ab 12D4 and humanized HB12B specifically recognized CD19 positivetumors.

FIG. 9 , comprising FIG. 9A and FIG. 9B, are a series of graphsdepicting the results of experiments illustrating that T cellselectroporated with Blina Bis-RNA alone were more sensitive anddisplayed longer persistence of anti-tumor activity than T cellselectroporated with CD19 CAR RNA. T cells electroporated with BlinaBis-RNA at 1 μg, 5 μg or 10 μg were compared with the T cellselectroporated with CD19BBZ (19OF) RNA at 10 μg. On multiple days postelectroporation, CD107a assay was conducted after those T cells werestimulated with CD19+cell lines (Naln6, K562-CD19 or Raji), or with CD19negative cell line K562 as control. FIG. 9A shows CD107a expression atday 3, 8 and 12 respectively. FIG. 9B shows both Mean FlouresenceIntensity (MFI) and percentage expression of CD107a at different dayspost electroporation. FIG. 9B demonstrates higher sensitivity and longerin vitro functional persistence of CD19-CD3 (Blina) RNA electroporated Tcells, comparing with CAR RNA T cells.

FIG. 10 is a series of graphs depicting the results of experimentsillustrating that T cells elctroporated with a Bis-RNA containing humanCD19 scFv (Blina-D4) function equally as well as T cells with BlinaBis-RNA. T cells electroporated with Blina-D4 Bis-RNA at 10 μg or 20 μg,or with 10 μg Blina Bis-RNA and subjected to CAR staining (Anti-mouseIgG Fab), upper panel (day 1 post electroporation) and Cd107a staining(lower panel).

FIG. 11 , comprising FIG. 11A and FIG. 11B, are a series of graphsdepicting the results of experiments illustrating that T cellselectroporated with Bis-RNAs against mesothelin, cMet or PSCA functionedequally as well as T cells electroporated with CAR RNA. T cellselectroporated with 10 μg Bis-RNA of ss1HL-Blina, or ss1LH-Blina, orcMet-Blina, or PSCA-Blina or GD2-Blina, were compared to T cellselectroporated with 10 μg ss1BBZ (ss1.OF), or cMetBBZ, or PSCA.BBZ orGD2.BBZ CAR RNA. FIG. 11A depicts results of CAR staining (Anti-mouseIgG Fab) of cells 1 day post electroporation (note that cMet CAR is ahuman origin scFv). FIG. 11B depicts results of Cd107a staining(CD8+gated).

DETAILED DESCRIPTION

The invention relates to compositions and methods for treating cancer,including, but not limited to, hematologic malignancies and solidtumors. The present invention relates to a strategy of adoptive celltransfer of T cells modified to express a bispecific antibody. Inanother embodiment, the invention relates to adoptive cell transfer of Tcells modified to express a chimeric antigen receptor (CAR).

In one embodiment, the T cells are modified to express a bispecificantibody. A bispecific antibody comprises two different bindingspecificities and thus binds to two different antigens. In oneembodiment, the bispecific antibody comprises a first antigenrecognition domain that binds to a first antigen and a second antigenrecognition domain that binds to a second antigen. In one embodiment,the first antigen recognition domain binds to a tumor associatedantigen. In one embodiment, the second antigen recognition region bindsto an antigen on T cells. In a particular embodiment, the second antigenrecognition region binds to CD3 on T cells. In one embodiment, theinvention relates to adoptive cell transfer of T cells modified toexpress a human or humanized bispecific antibody.

In one embodiment, the T cells are modified to express a CAR. CARs aremolecules that combine antibody-based specificity for a desired antigen(e.g., tumor antigen) with a T cell receptor-activating intracellulardomain to generate a chimeric protein that exhibits a specificanti-tumor cellular immune activity. In one embodiment, the inventionrelates to adoptive cell transfer of T cells modified to express a humanor humanized CAR.

In another embodiment, the invention relates to adoptive cell transferof T cells modified to express a bispecific antibody and a chimericantigen receptor (CAR).

The present invention relates generally to the use of T cellsgenetically modified to express a desired CAR, as well as to the use ofT cells genetically modified to express a desired CAR in combinationwith a desired bispecific antibody. T cells expressing a CAR arereferred to herein as CAR T cells or CAR modified T cells. Preferably,the cell can be genetically modified to express an antibody bindingdomain on its surface, conferring novel antigen specificity that is MHCindependent. In some instances, the T cell is genetically modified toexpress a CAR that combines an antigen recognition domain of a specificantibody with an intracellular domain of the CD3-zeta chain or FcyRIprotein into a single chimeric protein. In some instances, the CAR Tcell is genetically modified to express a CAR that combines an antigenrecognition domain of a specific antibody with an intracellular domainof the CD3-zeta chain or FcyRI protein into a single chimeric protein,as well as a bispecific antibody.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the CAR can comprise ahumanized antibody, or fragment thereof. In one embodiment, the CAR cancomprise a human antibody, or fragment thereof. In one embodiment, thetransmembrane domain that naturally is associated with one of thedomains in the CAR is used. In another embodiment, the transmembranedomain can be selected or modified by amino acid substitution to avoidbinding of such domains to the transmembrane domains of the same ordifferent surface membrane proteins to minimize interactions with othermembers of the receptor complex. In some embodiments, the extracellulardomain also comprises a hinge domain. Preferably, the hinge domaincomprises the CD8α hinge domain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise any desired cytoplasmic domain(s) useful in thecontext of the CAR of the invention. In one embodiment, the cytoplasmicdomain of the CAR can be designed to further comprise the signalingdomains of CD3-zeta, 4-1BB, and/or CD28. For example, the cytoplasmicdomain of the CAR can include but is not limited to CD3-zeta, 4-1BB andCD28 signaling modules and combinations thereof. Accordingly, theinvention provides CAR T cells and methods of their use for adoptivetherapy.

In one embodiment, the T cells are modified to express a bispecificantibody, in combination with a CAR. In one embodiment co-expression ofthe bispecific antibody and the CAR improves tumor recognition and tumorlysis.

In one embodiment, the modified T cells of the invention can begenerated by introducing a lentiviral vector comprising a desiredbispecific antibody into a T cell. In one embodiment, the modified Tcells of the invention can be generated by introducing a lentiviralvector comprising a desired CAR, for example a CAR comprising anti-CD19,CD8α hinge, and CD3zeta signaling domains into a T cell. In oneembodiment, the modified T cells of the invention can be generated byintroducing a lentiviral vector comprising a desired bispecific antibodyin combination with a lentivirus vector comprising a desired CAR into aT cell. In another embodiment, the modified T cells of the invention canbe generated by introducing a lentiviral vector comprising a desiredCAR, for example a CAR comprising anti-CD19, CD8α hinge, and CD3zetasignaling domains, and a desired bispecific antibody into a T cell. Inone embodiment, the modified T cells of the invention are able toreplicate in vivo resulting in long-term persistence that can lead tosustained tumor control.

In one embodiment, the modified T cells of the invention can begenerated by transfecting an RNA encoding a desired bispecific antibodyinto a T cell. In one embodiment, the modified T cells of the inventioncan be generated by transfecting an RNA encoding a desired CAR, forexample a CAR comprising anti-CD19, CD8α hinge, and CD3zeta signalingdomains into a T cell. In one embodiment, the modified T cells of theinvention can be generated by transfecting an RNA encoding a desiredbispecific antibody in combination with an RNA encoding the desired CARinto a T cell. In another embodiment, the modified T cells of theinvention can be generated by transfecting an RNA encoding the desiredCAR, for example a CAR comprising anti-CD19, CD8α hinge, and CD3zetasignaling domains, and further encoding a desired bispecific antibodyinto the cells. In one embodiment, both the CAR and the bispecificantibody are transiently expressed in the genetically modified T cells.

In one embodiment, the modified T cells of the invention can begenerated by transfecting an RNA encoding the desired CAR, for example aCAR comprising anti-CD19, CD8α hinge, and CD3zeta signaling domains intoa T cell, and introducing a lentiviral vector comprising the desiredbispecific antibody. In another embodiment, the modified T cells of theinvention can be generated by introducing a lentiviral vector comprisinga desired CAR, for example a CAR comprising anti-CD19, CD8α hinge, andCD3zeta signaling domains into a T cell, and transfecting an RNAencoding a desired bispecific antibody into the cell.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a humanized bispecific antibody, a humanizedCAR, or combination thereof for the treatment of a patient having canceror at risk of having cancer using lymphocyte infusion. In anotherembodiment the invention relates to administering a genetically modifiedT cell expressing a bispecific antibody, a CAR, or combination thereoffor the treatment of a patient having cancer or at risk of having cancerusing lymphocyte infusion. Preferably, autologous lymphocyte infusion isused in the treatment. Autologous PBMCs are collected from a patient inneed of treatment and T cells are activated and expanded using themethods described herein and known in the art and then infused back intothe patient.

In one embodiment, the invention relates to a CAR comprising human orhumanized antibodies, or fragments thereof. In one embodiment, theinvention relates to a bispecific antibody comprising human or humanizedantibodies, or fragments thereof. The invention is based upon thediscovery that constructs derived from human or humanized antibodiesspecifically recognize tumor antigens. Therefore, such human orhumanized constructs can be used to treat cancers and other disordersand avoid the risk of inducing an immune response.

In one embodiment, the invention relates to genetically modified T cellsexpressing a CAR and a bispecific antibody. The present invention isbased upon the finding that co-expression of a CAR and a bispecificantibody significantly enhanced tumor reactivity and tumor lysis.Further, induction of T cells to produce a CAR and a bispecific antibodyrecruits non-reactive T cells to become tumor reactive. This allows amethod to localize the delivery of anti-tumor agents to the specifictumor microenvironment using T cells. In one embodiment, CAR T cellstraffic the bispecific antibody to the site of the tumor, therebyreducing the toxicity associated with systemic delivery of bispecificantibodies.

In one embodiment, the invention relates to genetically modified T cellsexpressing a bispecific antibody. The present invention is partly basedon the finding that a T cell modified to express a bispecific antibodyperforms equally as well as a T cell modified to express a CAR.

In yet another embodiment, the invention relates generally to thetreatment of a patient at risk of developing cancer. The invention alsoincludes treating a malignancy or an autoimmune disease in whichchemotherapy and/or immunotherapy in a patient results in significantimmunosuppression in the patient, thereby increasing the risk of thepatient of developing cancer.

The invention includes using T cells expressing an anti-CD19 CAR and abispecific antibody. In one embodiment, T cells expressing the anti-CD19CAR and bispecific antibody of the invention display enhanced tumorrecognition and tumor lytic activity compared to T cells expressinganti-CD19 CAR alone. In some instances, the modified T cells of theinvention infused into a patient can eliminate cancerous cells in vivoin patients with cancer. However, the invention is not limited toCAR-expressing T cells. Rather, the invention includes any antigenbinding domain fused with one or more intracellular domains selectedfrom the group of a CD137 (4-1BB) signaling domain, a CD28 signalingdomain, a CD3zeta signal domain, and any combination thereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of±20% or ±10%, more preferably±5%, even more preferably±1%,and still more preferably±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird etal., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

A “bispecific antibody,” as used herein, refers to an antibody havingbinding specificities for at least two different antigenic epitopes. Inone embodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs. See,e.g., Milstein et al. (1983) Nature 305:537-39. Alternatively,bispecific antibodies can be prepared using chemical linkage. See, e.g.,Brennan et al. (1985) Science 229:81. Bispecific antibodies includebispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber et al. (1994) J. Immunol.152:5368.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject,” “patient” and “individual” are used interchangeablyherein and are intended to include living organisms in which an immuneresponse can be elicited (e.g., mammals). Examples of subjects includehumans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods for treatingcancer as well as other diseases. The cancer may be a hematologicalmalignancy, a solid tumor, a primary or a metastasizing tumor. Otherdiseases treatable using the compositions and methods of the inventioninclude viral, bacterial and parasitic infections as well as autoimmunediseases.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR wherein the CAR T cell exhibits an antitumorproperty. In a preferred embodiment, the CAR is a humanized CAR. The CARof the invention can be engineered to comprise an extracellular domainhaving an antigen binding domain fused to an intracellular signalingdomain of the T cell antigen receptor complex zeta chain (e.g., CD3zeta). The CAR of the invention when expressed in a T cell is able toredirect antigen recognition based on the antigen binding specificity.An exemplary antigen is CD19 because this antigen is expressed on B celllymphomas. However, the invention is not limited to targeting CD19.Rather, the invention includes any antigen binding domain that whenbound to its cognate antigen, affects a tumor cell so that the tumorcell fails to grow, is prompted to die, or otherwise is affected so thatthe tumor burden in a patient is diminished or eliminated. The antigenbinding domain is preferably fused with an intracellular domain from oneor more of a costimulatory molecule and a zeta chain. Preferably, theantigen binding domain is fused with one or more intracellular domainsselected from the group of a CD137 (4-1BB) signaling domain, a CD28signaling domain, a CD3zeta signal domain, and any combination thereof.

In one embodiment, the invention provides a T cell engineered to expressa bispecific antibody. In one embodiment, the bispecific antibodycomprises a human antibody, or fragment thereof. In one embodiment, thebispecific antibody comprises a humanized antibody, or fragment thereof.The bispecific antibody comprises two different binding specificities.In one embodiment, the bispecific antibody comprises a region that bindsto a tumor antigen. In one embodiment, the bispecific antibody comprisesa region that binds to a T cell antigen. For example, in one embodiment,the bispecific antibody binds to CD3.

In one embodiment, the invention provides a T cell engineered to expressa CAR and a bispecific antibody. In some embodiments, the CAR is ahumanized CAR. In some embodiments, the bispecific antibody recognizesthe same antigen as recognized by the CAR. In other embodiments, thebispecific antibody recognizes a different antigen. The presentinvention is based upon the discovery that T cells modified to expressboth a CAR and a bispecific antibody display enhanced tumor recognitionand tumor lytic activity. Further, co-expression or co-introduction of aCAR and a bispecific antibody allows non-reactive T cells to becometumor reactive. Thus, the present invention provides the specificdelivery of the bispecific antibody to a tumor microenvironment, therebyalleviating toxicity associated with systemic delivery of bispecificantibodies.

In some embodiments, the present invention is directed to retroviral orlentiviral vectors encoding a CAR and/or bispecific antibody that isstably integrated into a T cell and stably expressed therein. In otherembodiments, the present invention is directed to RNA encoding a CARand/or a bispecific antibody that is transfected into a T cell andtransiently expressed therein. Transient, non-integrated expression ofthe CAR and bispecific antibody in a cell mitigates concerns associatedwith permanent and integrated expression in a cell.

In some embodiment, the present invention includes introducing abispecific antibody along with a CD19 CAR in order to enhance theanti-tumor activity of the CAR engineered T cell. In one embodiment, thebispecific antibody is in the form of RNA. In another embodiment, theCD19 CAR is in the form of RNA. In yet another embodiment, thebispecific antibody and CD19 CAR are in the form of RNA.

In one embodiment, introducing bispecific antibody RNA along with CD19CARs enhances the anti-tumor activity of CAR engineered T cells.Further, introduction of bispecific antibody RNA into T cells recruitsnon-tumor reactive T cells to become tumor reactive, which provides anovel way of delivering and trafficking an antitumor drug into cancerpatients by using T cells. This reduces the toxicity of systemic BiTEs,by focusing the delivery of the bispecific antibody to the tumormicroenvironment by virtue of the CAR T cell that carries the cargo(BiTE) to the site of the tumor.

Compositions

The present invention provides a chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. In someembodiments, the CAR of the invention is humanized. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding domain. In some embodiments, the extracellulardomain also comprises a hinge domain. The intracellular domain orotherwise the cytoplasmic domain comprises a costimulatory signalingregion and a zeta chain portion. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by poly A addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the gene to be expressed,and a poly A tail, typically 50-2000 bases in length. RNA so producedcan efficiently transfect different kinds of cells. In one embodiment,the template includes sequences for the CAR.

Preferably, the CAR comprises an extracellular domain, a transmembranedomain and a cytoplasmic domain. The extracellular domain andtransmembrane domain can be derived from any desired source of suchdomains. In some instances, the hinge domain of the CAR of the inventioncomprises the CD8α hinge domain. In one embodiment, the CAR comprisesthe nucleic acid sequence of any one of SEQ ID NOs: 20-23. In oneembodiment, the CAR comprises the amino acid sequence encoded by thenucleic acid sequence of any one of SEQ ID NOs: 20-23.

Antigen Binding Domain

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen of interest by way of engineering a desired antigenbinding domain that specifically binds to an antigen on a tumor cell. Inthe context of the present invention, “tumor antigen” or“hyperporoliferative disorder antigen” or “antigen associated with ahyperproliferative disorder,” refers to antigens that are common tospecific hyperproliferative disorders such as cancer. The antigensdiscussed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding domain of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding domain portion of the CARtargets an antigen that includes but is not limited to CD19, CD20, CD22,ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII,GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifCD19 is the desired antigen that is to be targeted, an antibody for CD19can be used as the antigen bind moiety for incorporation into the CAR ofthe invention. In one embodiment, the antigen binding domain portion ofthe CAR of the invention targets CD19.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to monoclonal antibodies, polyclonalantibodies, synthetic antibodies, human antibodies, humanizedantibodies, and fragments thereof. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise a human antibody or fragment thereof. Thus, in oneembodiment, the antigen biding domain portion comprises a human antibodyor a fragment thereof. For example, the antigen binding domain of SEQ IDNO: 20 is fully human in origin.

For in vivo use of antibodies in humans, it may be preferable to usehuman antibodies. Completely human antibodies are particularly desirablefor therapeutic treatment of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods using antibody libraries derived from human immunoglobulinsequences, including improvements to these techniques. See, also, U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO91/10741; each of which is incorporated herein by reference in itsentirety. A human antibody can also be an antibody wherein the heavy andlight chains are encoded by a nucleotide sequence derived from one ormore sources of human DNA.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Anti-CD19 antibodies directed against thehuman CD19 antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies, including, butnot limited to, IgG1 (gamma 1) and IgG3. For an overview of thistechnology for producing human antibodies, see, Lonberg and Huszar (Int.Rev. Immunol., 13:65-93 (1995)). For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; and 5,939,598, each of which is incorporated byreference herein in their entirety. In addition, companies such asAbgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above. For a specificdiscussion of transfer of a human germ-line immunoglobulin gene array ingerm-line mutant mice that will result in the production of humanantibodies upon antigen challenge see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993);and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991)l Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al., Nature,348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of unimmunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905,each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Alternatively, in some embodiments, a non-human antibody is humanized,where specific sequences or regions of the antibody are modified toincrease similarity to an antibody naturally produced in a human. In oneembodiment, the antigen binding domain portion is humanized. Forexample, the antigen binding domain of SEQ ID NO: 21 is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5);489-498; Studnickaet al., 1994, Protein Engineering, 7(6);805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886,International Publication No. WO 9317105, Tan et al., J. Immunol.,169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000),Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem.,272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904(1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995),Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene,150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73(1994), each of which is incorporated herein in its entirety byreference. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well-known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeven et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

Antibodies can be humanized with retention of high affinity for thetarget antigen and other favorable biological properties. According toone aspect of the invention, humanized antibodies are prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind the target antigen.In this way, FR residues can be selected and combined from the recipientand import sequences so that the desired antibody characteristic, suchas increased affinity for the target antigen, is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding.

A “humanized” antibody retains a similar antigenic specificity as theoriginal antibody, i.e., in the present invention, the ability to bindhuman CD19 antigen. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody for human CD19antigen may be increased using methods of “directed evolution,” asdescribed by Wu et al., J. Mol. Biol., 294:151 (1999), the contents ofwhich are incorporated herein by reference herein in their entirety.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CD5,CD22, CD79a, CD79b, and CD66d. It is particularly preferred thatcytoplasmic signaling molecule in the CAR of the invention comprises acytoplasmic signaling sequence derived from CD3 zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3-zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention is exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta. In another embodiment, the cytoplasmicdomain is designed to comprise the signaling domain of CD3-zeta and thesignaling domain of 4-1BB.

Bispecific Antibodies

The present invention also provides a bispecific antibody. A bispecificantibody comprises two different binding specificities and thus binds totwo different antigens. In one embodiment, the bispecific antibodycomprises a first antigen recognition domain that binds to a firstantigen and a second antigen recognition domain that binds to a secondantigen. In one embodiment, the first antigen recognition domain bindsto a tumor associated antigen. As described elsewhere herein, thebispecific antibody can recognize the same or different tumor antigenrecognized by the CAR of the invention. In one embodiment, the secondantigen recognition region binds to a T cell antigen. For example, thebispecific antibody can recognize CD3. In some instances, a bispecificantibody that recognizes a T cell antigen is referred to as a BispecificT Cell Engager (BiTE). An exemplary BiTE is Blinatumomab (obtainablefrom Amgen) which comprises an anti-CD19 domain and an anti-CD3 domain.Blinatumomab (Blina) is encoded by the nucleic acid sequence of SEQ IDNO: 1. However, the present invention is not limited by the use of anyparticular bispecific antibody. Rather, any bispecific antibody or BiTEcan be used. Examples of tumor associated antigens are describedelsewhere herein, all of which may be targeted by the bispecificantibody of the present invention. In one embodiment, the bispecificantibody comprises a human antibody, a humanized antibody, or fragmentsthereof. Techniques for making human and humanized antibodies aredescribed elsewhere herein.

Techniques for making bispecific antibodies include, but are not limitedto, recombinant co-expression of two immunoglobulin heavy chain-lightchain pairs having different specificities (see Milstein and Cuello,Nature 305:537 (1983), WO 93/08829, and Traunecker et al., EMBO J.10:3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat.No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science 229:81 (1985)); using leucine zippers to producebispecific antibodies (see, e.g., Kostelny et al., J. Immunol.148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147:60 (1991). Engineered antibodies with three or morefunctional antigen binding sites, including “Octopus antibodies,” arealso included herein (see, e.g. US 2006/0025576A1). Bispecificantibodies can be constructed by linking two different antibodies, orportions thereof. For example, a bispecific antibody can comprise Fab,F(ab′)₂, Fab′, scFv, and sdAb from two different antibodies.

Vectors

The present invention encompasses a nucleic acid construct comprisingsequences of a CAR, wherein the sequence comprises the nucleic acidsequence of an antigen binding domain operably linked to the nucleicacid sequence of an intracellular domain. An exemplary intracellulardomain that can be used in the CAR of the invention includes but is notlimited to the intracellular domain of CD3-zeta, CD28, 4-1BB, and thelike. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, and the like. The present invention alsoencompasses a nucleic acid construct comprising sequences of bispecificantibody. The present invention further encompasses a nucleic acidconstruct comprising sequences of a CAR, wherein the sequence comprisesthe nucleic acid sequence of an antigen binding domain operably linkedto the nucleic acid sequence of an intracellular domain, and wherein thenucleic acid construct also comprises the nucleic acid sequence of abispecific antibody.

In one embodiment, the CAR of the invention comprises anti-CD19 antigenbinding domain, human CD8 hinge, and CD3zeta signaling domains. In oneembodiment, the CAR of the invention comprises the nucleic acid sequenceset forth in one of SEQ ID NOs: 20-23.

In one embodiment, the bispecific antibody of the antibody comprises thenucleic acid sequence set forth in one of SEQ ID NOs: 1-19.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a nucleic acid ofthe present invention is inserted. Vectors derived from retrovirusessuch as the lentivirus are suitable tools to achieve long-term genetransfer since they allow long-term, stable integration of a transgeneand its propagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See. e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno- associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the nucleic acid has been introduced into therecipient cells. Suitable reporter genes may include genes encodingluciferase, beta-galactosidase, chloramphenicol acetyl transferase,secreted alkaline phosphatase, or the green fluorescent protein gene(e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter- driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K &K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,AL). Stock solutions of lipids in chloroform or chloroform/methanol canbe stored at about −20° C. Chloroform is used as the only solvent sinceit is more readily evaporated than methanol. “Liposome” is a genericterm encompassing a variety of single and multilamellar lipid vehiclesformed by the generation of enclosed lipid bilayers or aggregates.Liposomes can be characterized as having vesicular structures with aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology5:505-10). However, compositions that have different structures insolution than the normal vesicular structure are also encompassed. Forexample, the lipids may assume a micellar structure or merely exist asnonuniform aggregates of lipid molecules. Also contemplated arelipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

RNA Transfection

In one embodiment, the genetically modified T cells of the invention aremodified through the introduction of RNA. In one embodiment, an in vitrotranscribed RNA CAR can be introduced to a cell as a form of transienttransfection. In another embodiment, the RNA CAR is introduced alongwith an in vitro transcribed RNA encoding a bispecific antibody. The RNAis produced by in vitro transcription using a polymerase chain reaction(PCR)-generated template. DNA of interest from any source can bedirectly converted by PCR into a template for in vitro mRNA synthesisusing appropriate primers and RNA polymerase. The source of the DNA canbe, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, syntheticDNA sequence or any other appropriate source of DNA. The desiredtemplate for in vitro transcription is the CAR and/or bispecificantibody of the present invention. By way of example, the templatecomprises an extracellular domain comprising a single chain variabledomain of an anti-tumor antibody; a transmembrane domain comprising thehinge and transmembrane domain of CD8a; and a cytoplasmic domaincomprises the signaling domain of CD3-zeta. By way of another example,the template comprises a bispecific antibody. By way of another example,the template comprises an extracellular domain comprising a single chainvariable domain of an anti-tumor antibody; a transmembrane domaincomprising the hinge and transmembrane domain of CD8a; and a cytoplasmicdomain comprises the signaling domain of CD3-zeta, and a bispecificantibody. In one embodiment, the template for the RNA-CAR is one of SEQID NO: 20-23. In one embodiment, the template for an RNA encoding abispecific antibody is one of SEQ ID NO: 1-19.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, it is not desirable to have prolonged ongoing stimulationof the immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly (A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of poly A/T stretches into a DNAtemplate is molecular cloning. However poly A/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with poly A/T 3′stretch without cloning highly desirable.

The poly A/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly (A) polymerase, such as E. colipoly A polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposornemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified T Cells

In some embodiments, the CAR sequences and/or bispecific antibodysequences are delivered into cells using a retroviral or lentiviralvector. CAR-expressing and/or bispecific antibody expressing retroviraland lentiviral vectors can be delivered into different types ofeukaryotic cells as well as into tissues and whole organisms usingtransduced cells as carriers or cell-free local or systemic delivery ofencapsulated, bound or naked vectors. The method used can be for anypurpose where stable expression is required or sufficient.

In other embodiments, the CAR sequences and/or bispecific antibodysequences are delivered into cells using in vitro transcribed mRNA. Invitro transcribed mRNA CAR can be delivered into different types ofeukaryotic cells as well as into tissues and whole organisms usingtransfected cells as carriers or cell-free local or systemic delivery ofencapsulated, bound or naked mRNA. The method used can be for anypurpose where transient expression is required or sufficient.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the chimeric receptor mRNAs with differentstructures and combination of their domains. For example, varying ofdifferent intracellular effector/costimulator domains on multiplechimeric receptors in the same cell allows determination of thestructure of the receptor combinations which assess the highest level ofcytotoxicity against multi-antigenic targets, and at the same timelowest cytotoxicity toward normal cells.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free: An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation.Preferably, it is desirable to stabilize IVT-RNA using variousmodifications in order to achieve prolonged expression of transferredIVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° ° C.or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺T cells or CD8⁺T cells, an anti-CD3 antibody and an anti-CD28antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28(Diaclone, Besançon, France) can be used as can other methods commonlyknown in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998;Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J.Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺T cell expansion and Tcell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1: 1, 2: 1, 3: 1, 4: 1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one preferred ratio being at least 1: 1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) modified toexpress a bispecific antibody, a CAR, or a combination thereof, wherethe CAR combines an antigen recognition domain of a specific antibodywith an intracellular domain of CD3-zeta, CD28, 4-1BB, or anycombinations thereof. Therefore, in some instances, the transduced Tcell can elicit a CAR-mediated T-cell response.

In one embodiment, the invention provides the use of a CAR to redirectthe specificity of a primary T cell to a tumor antigen. Thus, thepresent invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a T cell thatexpresses a CAR, wherein the CAR comprises a binding moiety thatspecifically interacts with a predetermined target, a zeta chain portioncomprising for example the intracellular domain of human CD3-zeta, and acostimulatory signaling region.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a bispecificantibody, a CAR, or combination thereof, and the T cell is infused to arecipient in need thereof. The infused cell is able to kill tumor cellsin the recipient. Unlike antibody therapies, in some embodiments themodified T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the T cells of the invention can undergo robust invivo T cell expansion and can persist for an extended amount of time. Inanother embodiment, the T cells of the invention evolve into specificmemory T cells that can be reactivated to inhibit any additional tumorformation or growth.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the -modified T cells may be an active ora passive immune response. In addition, the mediated immune response maybe part of an adoptive immunotherapy approach in which -modified T cellsinduce an immune response specific to the targeted antigen recognized bythe CAR and/or bispecific antibody.

In one embodiment, modified T cells of the invention secrete thebispecific antibody into the extracellular space of the tumormicroenvironment. This effective delivery method reduces the toxicityassociated with systemic delivery of bispecific antibodies.

In one embodiment, the present invention provides the use of a modifiedT cell to effectively deliver a bispecific antibody to a particularregion (i.e. a tumor microenvironment). In one embodiment, T cellsmodified to express a CAR and a bispecific antibody target a specifictumor microenvironment through the antigen binding domain of the CARexpressed on the surface of the CAR. Further, in one embodiment, theCAR-mediated delivery of bispecific antibodies arms non-modified T cellsof the tumor microenvironment with the therapeutic bispecific antibody.In one embodiment, the bispecific antibody binds to a T cell and a tumorantigen. This form of bispecific antibody, known as a BiTE, workstogether with the CAR to specifically recognize and kill tumors. In oneembodiment, the methods of the present invention comprise administeringT cells modified to express both a CAR and a bispecific antibody to asubject to enhance tumor recognition, immune response, and tumor lysis,compared to delivery of T cells modified with only a CAR.

While the data disclosed herein specifically described IVT RNA CARcomprising an anti-CD19 region, a human CD8α hinge and transmembraneregion, and 4-1BB and CD3zeta signaling domains, the invention should beconstrued to include any number of variations for each of the componentsof the construct as described elsewhere herein. That is, the inventionincludes the use of any antigen binding domain in the CAR to generate aCAR-mediated T-cell response specific to the antigen binding domain. Forexample, the antigen binding domain in the CAR of the invention cantarget a tumor antigen for the purposes of treat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the CAR and bispecific antibody of the invention aredesigned to treat a particular cancer. For example, the CAR andbispecific antibody designed to target CD19 can be used to treat cancersand disorders including but are not limited to pre-B ALL (pediatricindication), adult ALL, mantle cell lymphoma, diffuse large B-celllymphoma, salvage post allogenic bone marrow transplantation, and thelike.

In another embodiment, the CAR and bispecific antibody can be designedto target CD22 to treat diffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, the CAR and bispecific antibody can be designed totarget mesothelin to treat mesothelioma, pancreatic cancer, ovariancancer, and the like. In another embodiment, the CAR and bispecificantibody can be designed to target CD33/IL3Ra to treat acute myelogenousleukemia and the like. In a further embodiment, the CAR and bispecificantibody can be designed to target c-Met to treat triple negative breastcancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR and bispecific antibody can be designed totarget PSMA to treat prostate cancer and the like. In anotherembodiment, the CAR and bispecific antibody can be designed to targetGlycolipid F77 to treat prostate cancer and the like. In a furtherembodiment, the CAR and bispecific antibody can be designed to targetEGFRvIII to treat gliobastoma and the like.

In one embodiment, the CAR and bispecific antibody can be designed totarget GD-2 to treat neuroblastoma, melanoma, and the like. In anotherembodiment, the CAR and bispecific antibody can be designed to targetNY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, and the like. In afurther embodiment, the CAR and bispecific antibody can be designed totarget MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR and/or bispecific antibody can beused to treat the disease.

The modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CARand/or bispecific antibody to the cells, and/or iii) cryopreservation ofthe cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing compositions disclosed herein. Themodified cell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and themodified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as Flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the modified Tcells of the invention are used in the treatment of cancer. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing cancer. Thus, the present inventionprovides methods for the treatment or prevention of cancer comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the modified T cells of the invention.

The modified T cells of the present invention may be administered eitheralone, or as a pharmaceutical composition in combination with diluentsand/or with other components such as IL-2 or other cytokines or cellpopulations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Enhancing Anti-Tumor Activity of RNA CAR T Cells byCo-Introducing Bispecific Antibody RNA (Bis-RNA)

Treating cancer patients with adoptive transfer of CAR re-directed Tcells has shown promising results. Electroporation of RNA encoding CARsinto T cells provides a safer, easier and probably more efficientalternative way to currently commonly used lenti- or retro-viral basedgene deliver based therapies. The findings from recent clinical trialsdemonstrated that CARs derived from mouse Abs showed evidence of humananti-mouse antibody (HAMA) response, which potentially jeopardized thetherapy. Thus, CARs derived from human or humanized Abs provide adesirable alternative to mouse derived sequences, especially whenrepeated infusions are needed by using RNA electroporated T cells totreat cancer patients.

The studies disclosed herein describe: 1) the generation of anti-humanCD19 CARs derived from human origin that eliminate potential HAMA whenusing CARs by either lentiviral transduction or RNA electroporation: 2)the co-introduction of a gene (or mRNA) encoding a bispecific Ab derivedfrom human (or humanized) Abs. Here exemplary CARs were constructed(FIG. 1 ) with 4-1BB-zeta signaling domains against human CD19 fromanti-CD19 Abs that is either fully human in origin (clone 21D4; U.S.Pat. App. No.: 2010/0104509 A1), or humanized (clone HB12B; U.S. Pat.App. No.: US2008/0138336 A1) or mouse origin (CD19 scFv) fromBlinatumomab (anti-CD19/anti-CD3 bispecific Ab); U.S. Pat. No. 7,575,923B2). In addition to testing CD19-CD3 bispecific Ab RNA (Bis-RNA),mesothelin (ss1)-CD3 (ss1HL-Blina and ss1-BlinaLH), cMet-CD3(cMet-Blina), PSCA-CD3 (PSCA-Blina) and GD2-CD3 (Gd2-Blina) Bis-RNAswere constructed. To humanize Blina Bis-RNA, constructs were made andboth murine CD19 and CD3 scFv were replaced by human or humanized scFvagainst CD19 (21D4) and CD3 (27H5 VL1, 27H5 VL2, 28F11, DIVHv5, DIVHv6and DIVHv7) respectively, which resulted 13 constructs: D4-Blina,D4-VL1, D4-VL2, D4-F11, D4-Hv5, D4-Hv6, D4-Hv7, Blina-VL1, Blina-VL2,Blina-F11, Blina-Hv5, Blina-Hv6 and Blina-Hv7.

IVT RNA generated from the constructs encoding these CD19 CARs waselectroporated into T cells. CAR expression and anti-tumor activity werecompared with CD19 CAR derived from FMC63 (currently used for CART19trials). The experimental design for the results presented herein isshown in FIG. 2 . The results showed that all the new CD19 CARs could beefficiently expressed. In spite of the slightly reduced expression ascompared with the FMC63 CAR, their anti-tumor activity assayed by CD107adetection was comparable to the control FMC63 CAR.

To examine whether co-introduction of a gene (or mRNA) encodinganti-CD19/anti-CD3 bispecific Ab further enhanced anti-tumor activity ofCAR engineered T cells, the gene encoding Blinatumomab (Blina) wassynthesized by PCR, which was then constructed into IVT vector pGEM.64Ato generate pGEM-Blina.64A. T cells were co-electroporated with the RNAencoding Blinatumomab (Blina) and with CD19 CAR RNA (FCM63 CAR), oralternatively were electroporated with Blina RNA alone, and werecompared with T cells electroporated with FCM63 CD19 CAR RNA alone.Cells were stained for the presence of mouse IgG, which showed that notonly did CD19 CAR RNA electroporated cells stain positive for CARexpression, but also the co-incubation of Blina Bis-RNA electroporatedcells with GFP-RNA electroporated cells resulted in positive staining ina majority of cells (FIG. 3 ). This data is consistent with theexplanation that the CD19-CD3 bispecific Ab secreted by Blinaelectroporated T cells could bind to CD3 of GFP electroporated T cells.

Electroporated cells were co-cultured with CD19 expressing cells (Nalm6,K562-CD19, and Raji) or with the CD19 negative cells (K562). A CD107adegranulation assay was performed, which showed that that Blina Bis-RNAalone could enable electroporated T cells to specifically detect tumoras efficiently as FMC63 CD19 RNA electroporated T cells (FIG. 4 ).Co-electroporation of Blina RNA with CD19 CAR RNA could further enhancetumor reactivity, as evidenced by the CD107a assay (FIG. 4).

Furthermore, when mixing T cells co-electroporated with both CD19 CARRNA and Blina RNA or Blina RNA alone with T cells electroporated withonly GFP, it was found that bispecific Ab secreted from Blina RNAelectroporated T cells could arm non-tumor recognizing GFP+T cells toefficiently recognize CD19+ tumors (FIG. 5 ).

Electroporated cells were examined for their lytic activity in aflow-based CTL assay using K562-CD19-CFSE/K562-meso-CMRA as targetcells. Co-electroporation of Blina RNA with CD19 CAR RNA could furtherenhance killing activity of the T-cells (FIG. 6 ). Further,co-incubation of Blina RNA electroporated cells with non-tumorrecognizing GFP+cells maintained killing activity compared to thenon-diluted counterparts, demonstrating that secreted bis-RNA could bindto GFP-RNA electroporated T cells to efficiently kill CD19+tumors, in anantigen specific way (FIG. 6 ).

The new CD19 CAR RNA constructs were evaluated by examining theirexpression 15 hours post electroporation by staining for either mouseIgG or human IgG, which showed that all constructs induced CARexpression (FIG. 7 ). When co-cultured with CD19+tumor lines (K562-CD19or Raji) or with a CD negative line (U266B1), T cells electroporatedwith the new constructs displayed ability to specifically recognize CD19positive tumors, as evidenced by the CD107a assay (FIG. 8 ).

T cells were electroporated with Blina Bis-RNA at 1 μg, 5 μg or 10 μgand were compared with the T cells electroporated with FCM63 CD19 CARRNA at 10 μg. A CD107a assay was performed after, electroporated cellswere stimulated with CD19+cells (Naln6, K562-CD19, or Raji), or withCD19- cell line (K562). It was found that T cells with lug Blina Bis-RNAfunctioned nearly as well as T cells with 10 μg CD19 CAR RNA. T cellswith 10 μg CD19 CAR RNA lost their function at day 8-10 afterelectroporation, which is similar as the T cells with 1 μg BlinaBis-RNA, while the T cells with 5 μg or 10 μg Blina Bis-RNA continued tofunction up to day 14 post electroporation (FIG. 9B).

To assess the functionality of human and humanized forms of Bis-RNA, Tcells were electroporated with Blina-D4 Bis-RNA, in which CD19 scFv fromBlina was replaced with 21D4 scFv. Cells were subjected to CAR stainingand CD107a staining, which demonstrated that cells electroporated withBlina-D4 functioned equally well as T cells with Blina Bis-RNA (FIG. 10).

Bis-RNA constructs specific for other antigen markers were designed andconstructed. Comparing with T cells expressing CAR RNA for mesothelin(ss1), cMet or PSCA, it was found that the T cells expressing ss1-Blina,cMet-Blina, or PSCA-Blina Bis-RNA alone could function equally as wellas T cells with CAR RNA. Moreover, T cells with ss1-Blina Bis-RNAsshowed more antigen specific T cell activation than T cells with ssl CARRNA (FIG. 11 ).

The results presented herein demonstrate that introducing bispecificantibody RNA along with CD19 CARs enhances the anti-tumor activity ofCAR engineered T cells. Further, introduction of bispecific antibody RNAinto T cells recruits non-tumor reactive T cells to become tumorreactive, which provides a novel way of delivering and trafficking anantitumor drug into cancer patients by using T cells. This could reducethe toxicity of systemic BiTEs, by focusing the delivery of thebispecific antibody to the tumor microenvironment by virtue of the CAR Tcell that will carry the cargo (BiTE) to the site of the tumor.

Blinatumomab ORF (SEQ ID NO: 1)

atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa Blina-D4(SEQ ID NO: 2)atgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaagggcggggggggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttctggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaa Ss1.HL.CD3 (SEQ ID NO: 3)atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttacgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagacgacgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaaggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa Ss1.LH.CD3 (SEQ ID NO: 4)Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatccgacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttacgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagacgacgcaacttattactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa cMet.CD3 (SEQ ID NO: 5)atgctgctgctggtgaccagcctgctgctgtgtgagctgccccaccccgcctttctgctgatccccgacatccagatgacccagagccccagcagcgtgagcgccagcgtgggcgaccgggtgaccatcacctgccgggccagccagggcatcaacacctggctggcctggtatcagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgaagagcggcgtgcccagccggtttagcggctctggctctggcgccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcaggccaacagcttccccctgacctttggcggcggaacaaaggtggagatcaagggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggccaggtgcagctggtgcagagcggagccgaggtgaagaagcctggcgcctccgtcaaggtgtcctgcgaggccagcggctacaccttcaccagctacggcttcagctgggtgcggcaggcaccaggccagggcctcgaatggatgggctggatcagcgccagcaacggcaacacctactacgcccagaagctgcagggcagggtcaccatgaccaccgacaccagcaccagcagcgcctacatggaactgcggagcctgagaagcgacgacaccgccgtgtactactgcgccagggtgtacgccgactacgccgattactggggccagggcaccctggtgaccgtgagcagcggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa PSCA.CD3(SEQ ID NO: 6)atggcgctaccggtgaccgcactcctgctgccactcgccctcctgctccacgccgcccgccccgatatccagctgacccaatcaccgtcgtccctgtctgcctccgtgggcgaccgggtgacgatcacctgtagtgcctcgagcagtgtacggttcatccactggtaccaacagaagcccggcaaggcaccaaagcggctgatctacgacaccagcaagctggcgtctggggtgcccagcaggttctcgggaagtggtagtggcacagacttcactctcaccatcagttcactccagccggaggactttgccacctactattgccagcagtggtcctcgtccccctttaccttcggccagggaacaaaggtggaaattaagggttcgacctccggggggggctccggtgggggctccggcggggggggctcatcggaggttcagctggtggagagcggcggcggcctggtgcagcccggcgggagtctgcggctgtcctgtgccgccagcggcttcaacatcaaggactactacattcactgggtgcggcaagccccaggcaagggtctggagtgggtggcttggattgaccctgaaaacggcgacactgagttcgtgccaaaattccaggggcgggcgaccatctccgccgacacctccaagaatacggcctacctgcagatgaactccctgcgcgccgaagacacagcggtctactactgcaagacagggggtttctggggccagggcaccctcgtgaccgtttcgagtgccgccggcggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa GD2-CD3 (SEQ ID NO: 7)atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgctctagagatattttgctgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagtcttgtacaccgtaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaagctcctgattcacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgttctcaaagtacacatgttcctccgctcacgttcggtgctgggaccaagctggagctgaaacgggctgatgctgcaccaactgtatccatcttcccaggctcgggcggtggtgggtcgggtggcgaggtgaagcttcagcagtctggacctagcctggtggagcctggcgcttcagtgatgatatcctgcaaggcttctggttcctcattcactggctacaacatgaactgggtgaggcagaacattggaaagagccttgaatggattggagctattgatccttactatggtggaactagctacaaccagaagttcaagggcagggccacattgactgtagacaaatcgtccagcacagcctacatgcacctcaagagcctgacatctgaggactctgcagtctattactgtgtaagcggaatggagtactggggtcaaggaacctcagtcaccgtctcctcagccaaaacgacacccccatcagtctatggaggtggtggatccgatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcagtcgaaggtggaagtggaggttctggtggaagtggaggttcaggtggagtcgacgacgccgccattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaacatcatcaccatcatcattaataa D4-27H5 VL1 (D4-VL1)(SEQ ID NO: 8)AtgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaagggcggggggggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatcccagGTG cag ctc gtg gag TCC ggc GGC ggc GTT GTC cag CCT GGC cgc TCG ctg cgcctg tca tgc GCC GCT TCG ggt ttc ACG ttc agg TCG TAC GGG atg cac tgg GTCAGG cag GCG CCG gga AAA GGC CTG gag tgg GTG GCT atc ATC tgg TACGAC ggc TCC aag aag AAT TAT GCT gac TCC GTC aag gga CGG ttc ACA atc tcgCGT GAT aac tcg aag aac acc CTC TAC CTG cag atg AAT TCC CTC AGA GCCGAA gac ACA GCC gtg TAT TAT tgc GCC AGG ggc ACC ggc tat aac tgg TTCGAT CCA tgg ggc cag GGG ACC ctg GTT acc GTC tcc tcc GGA GGG GGG GGTagt gag atc gtg ctg acc cag tcg CCT CGC ACC CTG tcc CTG tcc CCT GGG gagCGC gcc ACC CTC tcg tgc AGG GCA tcg cag TCC gtc agt TCC TCC TAT ctg GCCtgg tac CAG cag aaa CCT ggc cag GCA cca AGG CTG ctg ATC tac GGA GCT tccTCG AGG GCA ACC GGG ATC CCC gac AGA TTT tcc GGA agc GGA AGTGGC ACA gac ttc ACC ctg acc ATC AGT AGG CTT GAC CCC gaa GAT ttc GCCGTG tac TAT tgc cag cag tac GGC TCC TCC ccc atc acc ttc ggc cag GGC ACAAGA ctg gag atc aag cac CAT cac cac cac cac TAA TAA gcggccgcD4-27H5 VL2 (D4-VL2) (SEQ ID NO: 9)AtgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaaggggggggcgggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatccCAG GTA CAG ctg GTC GAG tcc GGT ggg GGC gtg GTC cag CCC ggc CGG tccCTG CGC CTG TCG tgc GCT GCC TCC ggc TTT ACC ttc CGG TCG TAT GGCatg CAT tgg gtg cgc cag GCC CCC GGG aag ggg ctg gag tgg GTC gcc ATT ATCtgg tac gat GGC TCC AAG aag aac TAC gct GAT TCG GTT aag ggc cgc TTC ACCatt AGT CGG GAT aat TCG aag aat ACG CTG TAT CTC CAG atg aac TCC ctgAGG GCC GAG gac act gcc GTG TAC TAC tgc gcc CGG GGC ACA GGC TATaac tgg ttc gat ccc tgg GGT cag ggc ACC CTA gtg ACC GTC TCG TCT GGG GGCGGA ggg TCA GAT att ctc atg ACA cag TCG ccc TCT AGT CTT tcc GCC tcc gtgGGG GAC CGC GTG acc atc ACA tgc AGA GCT TCC CAG GGG atc tcc TCTGCG CTG gcc tgg TAT cag cag aag ccc GGG aag GCA CCC AAG ctg CTC ATCTAT TAC GCT tct TCG CTG CAA AGT GGG gtg ccg TCC CGC TTC tcc gga AGCggc TCC ggc ACG GAC tac acc CTC acc atc tcc TCC CTG CAG CCT GAG GATttc GCC acc TAT tac tgc cag cag TAT tac TCC acg ctg ACC TTC GGA GGA GGCACG AAA GTG gag atc aag CAC cac cac cac cac cac TAA TAA gcggccgcD4-28F11 (D4-F11) (SEQ ID NO: 10)AtgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaagggcggggggggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatcccagGTC cag ctg GTT gag TCT GGT GGC GGA gtg gtc cag CCC ggc CGG TCT CTCCGC ctg TCC tgc GCC GCT tcc GGG ttc AAG TTC TCC ggc TAC gga atg cac tgggtg AGG cag GCG CCA GGT aag GGG CTC GAG tgg GTC GCG gtg ATA tggTAT gac GGT AGC aag aag tat tac gtg gac AGT GTG AAA ggc CGC TTT acc atcTCA CGC GAC AAT TCT aag aac acc ctg TAC CTC CAG atg aac TCC ctg CGCgct gag GAC ACG GCG GTG TAC tac TGC GCT AGG cag atg GGG tac tgg cacTTC gac CTT tgg ggc AGG GGT acc CTG GTG acc gtc TCA TCC GGC ggc ggcGGG TCT gag ATC GTT CTG ACC CAA AGT CCG gcc ACA ctg tcc CTC TCCCCA GGA gag cgc GCT ACG CTT AGC tgc CGC gcc tcc cag AGC GTG TCC tcctac ctg gca tgg TAT cag cag aag CCG GGG cag GCG CCT CGA ctg CTG atc tac gacgcc TCG aac CGC GCG ACA GGT atc ccc gcg CGC ttc AGC GGC TCC GGT TCGGGG ACT GAT ttc acc ctg ACC atc tcc TCC CTC GAG cct gag GAT ttc GCA gtgtac tac tgc cag cag aga TCC AAT tgg ccc CCC CTC acc ttc ggc GGG GGA acc aagGTG gag atc aag cac cac cac cac CAT cac TAATAA D4-DIVHv5 (D4-Hv5)(SEQ ID NO: 11)Atgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaaggggggggcgggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatcccaggtt cag ctg gtg cag tcc ggc gcc gag gtg aag aag ccg ggc gct tct gtg aag gtc agc tgt aaagcc agt ggc tac aca ttc acc agg tac act atg cac tgg gtg cgc cag gca ccc ggg cag ggg ctggaa tgg atc ggg tac atc aat cct tcc cgc ggt tat act aac tat aat caa aag ttc aaa gac cgc gtgaca att acg acc gat aag agt tca tcc acc gct tac tta cag atg aac tcc ctc aag aca gag gac accgcc gtg tac tac tgt gcc cgc tac tac gac gac cat tac tgc ctg gac tac tgg ggc cag ggg accacc gta acc gtc agt agt ggc ggg ggc ggc agt cag atc gtg ctg acc cag agt ccg gcg acc ctgagt ctg tct cct ggt gag cgc gca acg ctg acg tgc tca gcc tcc tcg agt gcc tct tat atg aac tggtac cag cag aag ccc ggc aag gcc cct aag cgc tgg atc tac gac acc tcg aag cta gct tcg ggcgtc ccc tcc cgg ttc tcg ggc tcg ggg tcg ggc acg gac tat tct ctg acc atc aac agt ctg gaggca gag gac gcc gca acc tac tac tgc cag cag tgg agt tcg aat cct ttc acg ttt ggg cag gggacc aag gtg gaa atc aaa cac cat cac cac cat cac TAA TAA gcggccgcD4-DIVHv6 (D4-Hv6) (SEQ ID NO: 12)AtgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaaggggggggcgggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatcccaggttcagctggtgcagtccggcgccgaggtgaagaagccgggcgcttctgtgaaggtcagctgtaaagccagtggctacacattcaccaggtacactatgcactgggtgcgccaggcacccgggcaggggctggaatggatcgggtacatcaatccttcccgcggttatactaactatGCtcaaaagttcCaagaccgcgtgacaattacgaccgataagagttcatccaccgcttacttacagatgaactccctcaagacagaggacaccgccgtgtactactgtgcccgctactacgacgaccattactgcctggactactggggccaggggaccaccgtaaccgtcagtagtggcgggggcggcagtcagatcgtgctgacccagagtccggcgaccctgagtctgtctcctggtgagcgcgcaacgctgacgtgctcagcctcctcgagtgcctcttatatgaactggtaccagcagaagcccggcaaggcccctaagcgctggatctacgacacctcgaagctagcttcgggcgtcccctcccggttctcgggctcggggtcgggcacggactattctctgaccatcaacagtctggaggcagaggacgccgcaacctactactgccagcagtggagttcgaatcctttcacgtttgggcaggggaccaaggtggaaatcaaacaccatcaccaccatcacTAATAAgcggccgc D4-DIVHv7 (D4-Hv7) (SEQ ID NO: 13)AtgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaaggggggggcgggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttcggaggtggtggatcccaggttcagctggtgcagtccggcgccgaggtgaagaagccgggcgcttctgtgaaggtcagctgtaaagccagtggctacacattcaccaggtacactatgcactgggtgcgccaggcacccgggcaggggctggaatggatcgggtacatcaatccttcccgcggttatactaactataatcaaaagGtcaaagaccgcTtgacaattacgaccgataagagttcatccaccgcttacttacagatgaactccctcaagacagaggacaccgccgtgtactactgtgcccgctactacgacgaccattactgcctggactactggggccaggggaccaccgtaaccgtcagtagtggcgggggcggcagtcagatcgtgctgacccagagtccggcgaccctgagtctgtctcctggtgagcgcgcaacgctgacgtgctcagcctcctcgagtgcctcttatatgaactggtaccagcagaagcccggcaaggcccctaagcgctggatctacgacacctcgaagctagcttcgggcgtcccctcccggttctcgggctcggggtcgggcacggactattctctgaccatcaacagtctggaggcagaggacgccgcaacctactactgccagcagtggagttcgaatcctttcacgtttgggcaggggaccaaggtggaaatcaaacaccatcaccaccatcacTAATAAgcggccgc Blina-27H VL1 (Blina-VL1) (SEQ ID NO: 14)atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatcccag GTG cag ctc gtg gag TCC ggc GGC ggc GTTGTC cag CCT GGC cgc TCG ctg cgc ctg tca tgc GCC GCT TCG ggt ttc ACG ttc aggTCG TAC GGG atg cac tgg GTC AGG cag GCG CCG gga AAA GGC CTG gag tggGTG GCT atc ATC tgg TAC GAC ggc TCC aag aag AAT TAT GCT gac TCC GTCaag gga CGG ttc ACA atc tcg CGT GAT aac tcg aag aac acc CTC TAC CTG cag atgAAT TCC CTC AGA GCC GAA gac ACA GCC gtg TAT TAT tgc GCC AGG ggcACC ggc tat aac tgg TTC GAT CCA tgg ggc cag GGG ACC ctg GTT acc GTC tcctcc GGA GGG GGG GGT agt gag atc gtg ctg acc cag tcg CCT CGC ACC CTG tccCTG tcc CCT GGG gag CGC gcc ACC CTC tcg tgc AGG GCA tcg cag TCC gtc agtTCC TCC TAT ctg GCC tgg tac CAG cag aaa CCT ggc cag GCA cca AGG CTG ctgATC tac GGA GCT tcc TCG AGG GCA ACC GGG ATC CCC gac AGA TTT tccGGA agc GGA AGT GGC ACA gac ttc ACC ctg acc ATC AGT AGG CTT GACCCC gaa GAT ttc GCC GTG tac TAT tgc cag cag tac GGC TCC TCC ccc atc acc ttcggc cag GGC ACA AGA ctg gag atc aag cac CAT cac cac cac cac TAA TAAgcggccgc Blina-27H VL2 (Blina-VL2) (SEQ ID NO: 15)atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatccCAG GTA CAG ctg GTC GAG tcc GGT ggg GGCgtg GTC cag CCC ggc CGG tcc CTG CGC CTG TCG tgc GCT GCC TCC ggc TTTACC ttc CGG TCG TAT GGC atg CAT tgg gtg cgc cag GCC CCC GGG aag ggg ctggag tgg GTC gcc ATT ATC tgg tac gat GGC TCC AAG aag aac TAC gct GAT TCGGTT aag ggc cgc TTC ACC att AGT CGG GAT aat TCG aag aat ACG CTG TATCTC CAG atg aac TCC ctg AGG GCC GAG gac act gcc GTG TAC TAC tgc gccCGG GGC ACA GGC TAT aac tgg ttc gat ccc tgg GGT cag ggc ACC CTA gtg ACCGTC TCG TCT GGG GGC GGA ggg TCA GAT att ctc atg ACA cag TCG ccc TCTAGT CTT tcc GCC tcc gtg GGG GAC CGC GTG acc atc ACA tgc AGA GCT TCCCAG GGG atc tcc TCT GCG CTG gcc tgg TAT cag cag aag ccc GGG aag GCACCC AAG ctg CTC ATC TAT TAC GCT tct TCG CTG CAA AGT GGG gtg ccgTCC CGC TTC tcc gga AGC ggc TCC ggc ACG GAC tac acc CTC acc atc tcc TCCCTG CAG CCT GAG GAT ttc GCC acc TAT tac tgc cag cag TAT tac TCC acg ctgACC TTC GGA GGA GGC ACG AAA GTG gag atc aag CAC cac cac cac cac cacTAA TAA gcggccgc Blina-28F11 (Blina-F11) (SEQ ID NO: 16)atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatcccag GTC cag ctg GTT gag TCT GGT GGC GGA gtggtc cag CCC ggc CGG TCT CTC CGC ctg TCC tgc GCC GCT tcc GGG ttc AAGTTC TCC ggc TAC gga atg cac tgg gtg AGG cag GCG CCA GGT aag GGG CTCGAG tgg GTC GCG gtg ATA tgg TAT gac GGT AGC aag aag tat tac gtg gac AGTGTG AAA ggc CGC TTT acc atc TCA CGC GAC AAT TCT aag aac acc ctg TACCTC CAG atg aac TCC ctg CGC gct gag GAC ACG GCG GTG TAC tac TGC GCTAGG cag atg GGG tac tgg cac TTC gac CTT tgg ggc AGG GGT acc CTG GTG accgtc TCA TCC GGC ggc ggc GGG TCT gag ATC GTT CTG ACC CAA AGT CCGgcc ACA ctg tcc CTC TCC CCA GGA gag cgc GCT ACG CTT AGC tgc CGC gcctcc cag AGC GTG TCC tcc tac ctg gca tgg TAT cag cag aag CCG GGG cag GCGCCT CGA ctg CTG atc tac gac gcc TCG aac CGC GCG ACA GGT atc ccc gcg CGCttc AGC GGC TCC GGT TCG GGG ACT GAT ttc acc ctg ACC atc tcc TCC CTCGAG cct gag GAT ttc GCA gtg tac tac tgc cag cag aga TCC AAT tgg ccc CCC CTCacc ttc ggc GGG GGA acc aag GTG gag atc aag cac cac cac cac CAT cac TAATAABlina-DIVHv5 (Blina-Hv5) (SEQ ID NO: 17)atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatcccag gtt cag ctg gtg cag tcc ggc gcc gag gtg aag aag ccgggc gct tct gtg aag gtc agc tgt aaa gcc agt ggc tac aca ttc acc agg tac act atg cac tgg gtgcgc cag gca ccc ggg cag ggg ctg gaa tgg atc ggg tac atc aat cct tcc cgc ggt tat act aactat aat caa aag ttc aaa gac cgc gtg aca att acg acc gat aag agt tca tcc acc gct tac tta cagatg aac tcc ctc aag aca gag gac acc gcc gtg tac tac tgt gcc cgc tac tac gac gac cat tactgc ctg gac tac tgg ggc cag ggg acc acc gta acc gtc agt agt ggc ggg ggc ggc agt cag atcgtg ctg acc cag agt ccg gcg acc ctg agt ctg tct cct ggt gag cgc gca acg ctg acg tgc tcagcc tcc tcg agt gcc tct tat atg aac tgg tac cag cag aag ccc ggc aag gcc cct aag cgc tggatc tac gac acc tcg aag cta gct tcg ggc gtc ccc tcc cgg ttc tcg ggc tcg ggg tcg ggc acggac tat tct ctg acc atc aac agt ctg gag gca gag gac gcc gca acc tac tac tgc cag cag tggagt tcg aat cct ttc acg ttt ggg cag ggg acc aag gtg gaa atc aaa cac cat cac cac cat cacTAA TAA gcggccgc Blina-DIVHv6 (Blina-Hv6) (SEQ ID NO: 18)atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatcccaggttcagctggtgcagtccggcgccgaggtgaagaagccgggcgcttctgtgaaggtcagctgtaaagccagtggctacacattcaccaggtacactatgcactgggtgcgccaggcacccgggcaggggctggaatggatcgggtacatcaatccttcccgcggttatactaactatGCtcaaaagttcCaagaccgcgtgacaattacgaccgataagagttcatccaccgcttacttacagatgaactccctcaagacagaggacaccgccgtgtactactgtgcccgctactacgacgaccattactgcctggactactggggccaggggaccaccgtaaccgtcagtagtggcgggggcggcagtcagatcgtgctgacccagagtccggcgaccctgagtctgtctcctggtgagcgcgcaacgctgacgtgctcagcctcctcgagtgcctcttatatgaactggtaccagcagaagcccggcaaggcccctaagcgctggatctacgacacctcgaagctagcttcgggcgtcccctcccggttctcgggctcggggtcgggcacggactattctctgaccatcaacagtctggaggcagaggacgccgcaacctactactgccagcagtggagttcgaatcctttcacgtttgggcaggggaccaaggtggaaatcaaacaccatcaccaccatcacTAATAAgcggccgcBlina-DIVHv7 (Blina-Hv7) (SEQ ID NO: 19):atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccggaggtggtggatcccaggttcagctggtgcagtccggcgccgaggtgaagaagccgggcgcttctgtgaaggtcagctgtaaagccagtggctacacattcaccaggtacactatgcactgggtgcgccaggcacccgggcaggggctggaatggatcgggtacatcaatccttcccgcggttatactaactataatcaaaagGtcaaagaccgcTtgacaattacgaccgataagagttcatccaccgcttacttacagatgaactccctcaagacagaggacaccgccgtgtactactgtgcccgctactacgacgaccattactgcctggactactggggccaggggaccaccgtaaccgtcagtagtggcgggggcggcagtcagatcgtgctgacccagagtccggcgaccctgagtctgtctcctggtgagcgcgcaacgctgacgtgctcagcctcctcgagtgcctcttatatgaactggtaccagcagaagcccggcaaggcccctaagcgctggatctacgacacctcgaagctagcttcgggcgtcccctcccggttctcgggctcggggtcgggcacggactattctctgaccatcaacagtctggaggcagaggacgccgcaacctactactgccagcagtggagttcgaatcctttcacgtttgggcaggggaccaaggtggaaatcaaacaccatcaccaccatcacTAATAAgcggccgc D4(19)-BBz ORF (SEQ ID NO: 20):atgggctggtcttgcatcatcctgttcctcgtggccaccgccaccggcgtccacagcgccatccagctcacccagagcccctcgagcttgagtgcctcggtgggagaccgggtcactatcacctgccgagccagtcagggcatctcctccgcccttgcctggtaccagcagaagcccgggaaggcccccaagctgctgatctacgacgctagtagtctggagagtggcgtgccttcgcgcttctcgggcagtgggagtggcaccgacttcaccttgaccatctccagtctacagccggaagatttcgcgacctactactgtcagcaattcaactcttatccatacactttcggccaggggacaaagctggagatcaaggggggggcgggagtggcggcggagggtccggaggcgggggctccgaggtgcaactagtccagagcggagccgaggtgaagaagcccggggagagtctaaagatctcttgcaagggctccggttactccttctcgagttcctggatcgggtgggtgcgacagatgccgggcaagggcctggagtggatgggcattatctaccccgacgactccgatacccgttatagtccatcgttccagggacaggtgaccatttccgccgacaagtctatcagaaccgcctatctgcagtggtccagtctgaaggcctctgacactgccatgtattattgcgccaggcacgttacgatgatctggggggtgatcatcgacttctggggccagggcacactcgtaaccgtcagttctgcggccgcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgacgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaataa 12B(19)-BBZ ORF (SEQ ID NO: 21):atggggtggtcgtgcatcatcctgtttctggtggccacagcaaccggcgtgcacagtgagattgtgctgacccaaagcccggacttccagtccgtgacccccaaggagaaggttaccatcacgtgccgcgcctctgaaagcgtggacacgttcgggatctccttcatgaattggtttcagcagaagccagatcagtcacccaaactcctgatccacgccgccagtaatcagggctcaggcgtcccgtccaggttctctggcagtggctccggtactgacttcaccttaaccatcaactctctggaggcagaggacgccgccacatacttctgccaacagagcaaggaggtgcccttcaccttcggaggtgggaccaaggtcgaaatcaagggagggggggggtccggcggcggcggatccggaggcggcggcagcgaggtgcagctcgtcgagagtgggggcggactggtgcaaccagggggctctctgcggctgagctgcgctgcctccggattcacattctcctcgtcctggatgaactgggttcgccaggcccccggcaaaggcctggagtgggtcggcagaatctacccaggcgacggggacacgaactacaacggcaagttcaagggccggttcacaatctcgcgcgacgactcaaaaaacagcctgtatctccagatgaactccctgaaaaccgaggacaccgccgtgtattactgtgcacgcagcggctttatcaccaccgttctggacttcgattattggggccagggtaccctggtgacggtaagttcggcggccgcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgacgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaataaBlina. 19BBZ ORF (SEQ ID NO: 22):atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactccgactacaaagatgatgacgataaggatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtggtggtggttctggcggcggcggctccggtggtggtggttctcaggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctcctccgcggccgcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgacgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaataa

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. An isolated nucleic acid molecule comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen binding domain derived from a bispecific antibody, atransmembrane domain, and a CD3 zeta signaling domain, further whereinthe antigen binding domain is selected from the group consisting of ahuman antibody, a humanized antibody, an antigen binding fragmentthereof, and any combination thereof.
 2. The isolated nucleic acidmolecule of claim 1, wherein the nucleic acid sequence comprises SEQ IDNO: 20, 21, or
 22. 3. The isolated nucleic acid sequence of claim 1,wherein the antigen-binding fragment is a Fab or a scFv.
 4. The isolatednucleic acid molecule of claim 1, wherein the antigen binding domainbinds to a tumor antigen.
 5. The isolated nucleic acid molecule of claim4, wherein the tumor antigen is associated with a hematologicmalignancy.
 6. The isolated nucleic acid molecule of claim 4, whereinthe tumor antigen is associated with a solid tumor.
 7. The isolatednucleic acid molecule of claim 4, wherein the tumor antigen is selectedfrom the group consisting of CD19, CD20, CD22, ROR1, mesothelin,CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR,MAGE A3 TCR, and any combination thereof.
 8. The isolated nucleic acidmolecule of claim 1, wherein the CAR further comprises a costimulatorysignaling region comprising an intracellular domain of a costimulatorymolecule selected from the group consisting of CD27, CD28, 4-1BB, OX40,CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,and any combination thereof.
 9. The isolated nucleic acid molecule ofclaim 1, wherein the nucleic acid sequence of the antigen binding domainderived from a bispecific antibody encodes a bispecific antibody. 10.The isolated nucleic acid molecule of claim 9, wherein the nucleic acidsequence encoding the bispecific antibody comprises the nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, and any combination thereof. 11-47.(canceled)
 48. An isolated nucleic acid molecule comprising: a firstnucleic acid sequence encoding a chimeric antigen receptor (CAR),wherein the CAR comprises one or more antigen binding domains of a firstbispecific antibody, a transmembrane domain, and a CD3 zeta signalingdomain, further wherein each antigen binding domain is selected from thegroup consisting of a human antibody, a humanized antibody, and anantigen binding fragment thereof; and a second nucleic acid sequenceencoding a second bispecific antibody, wherein the second bispecificantibody does not comprise and is not linked to a transmembrane domain.49. The isolated nucleic acid molecule of claim 48, wherein the secondbispecific antibody comprises a first antigen recognition domain thatrecognizes the same antigen as the antigen binding domain of the CAR,and a second antigen recognition domain that recognizes CD3.
 50. Theisolated nucleic acid molecule of claim 48, wherein the first nucleicacid sequence comprises the nucleic acid sequence of SEQ ID NO: 20, 21,or
 22. 51. The isolated nucleic acid molecule of claim 48, wherein atleast one antigen binding domain of the CAR: (a) is an antigen bindingfragment; or (b) binds to a tumor antigen.
 52. The isolated nucleic acidmolecule of claim 51, wherein the antigen binding fragment is a Fab or ascFv.
 53. The isolated nucleic acid molecule of claim 51, wherein thetumor antigen is associated with a hematologic malignancy or a solidtumor.
 54. The isolated nucleic acid molecule of claim 51, wherein thetumor antigen is selected from the group consisting of CD19, CD20, CD22,ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII,GD-2, NY-ESO-1, and MAGE A3.
 55. The isolated nucleic acid molecule ofclaim 48, wherein the isolated nucleic acid molecule is an RNA molecule.56. The isolated nucleic acid molecule of claim 48, wherein the CARfurther comprises a costimulatory signaling region comprising anintracellular domain of a costimulatory molecule selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,CD83, and any combination thereof.
 57. The isolated nucleic acidmolecule of claim 48, wherein the nucleic acid sequence encoding the oneor more antigen binding domains of the first bispecific antibodycomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.